Project Summary Back pain is a leading cause of disability in the United States with an increasing prevalence in the aging population. One known cause of chronic back pain is intervertebral disc (IVD) degeneration. The disc, which separates vertebrae and is pivotal to mobility, is a composite cartilaginous tissue. The inner, proteoglycan-rich nucleus pulposis (NP) is confined at its periphery by the annulus fibrosus (AF). In healthy individuals, osmotic swelling in the NP, which can exceed several atmospheres of pressure, maintains vertebral body separation and translates stresses and strains to the AF. However, in disc degeneration the osmotic swelling of the NP is abolished, the disc space collapses, and the prestresses translated to the AF fall, but it is currently unknown what role this loss of prestress plays in dictating the mechanical microenvironment and homeostasis of endogenous cells. In this proposal, we hypothesize that this evolving mechanical microenvironment of the AF in the context of disc degeneration alters the homeostasis and phenotype of cells. Additionally, we hypothesize that intervention to restore NP pressurization can restore prestress to the AF to promote tensional homeostasis of the resident cells. The objective of this proposal is to understand the interplay between whole disc mechanics and degeneration, specifically how degeneration and treatment alters the prestressed mechanical microenvironment in the AF, changes how mechanical signals are transduced by AF cells, and results in aberrant remodeling of the AF. Additionally this work will test the hypothesis that NP repressurization can restore the prestressed environment of the AF. Specifically, I will explore the underlying hypothesis with three Aims: 1) determine the relationship between macroscale tissue deformation and local micro-mechanics of the AF in intact, depressurized, and respressurized discs, 2) determine the role of NP pressurization, depressurization, and repressurization in the transmission of strain to the cell-scale, and regulation of AF cell signaling, and 3) identify the role of an evolving pre-stressed microenvironment on mechanical signaling by AF cells. These aims will be accomplished using novel techniques including local micro-indentation, multiphoton elastography, and tissue engineering approaches utilizing electrospun fibrous scaffolds. Together, the findings from the proposed work will reveal the mechanical and biological interplay in disc degeneration. Specifically, this work will assess how altered cellular microenvironments evolve over the course of degeneration and how this evolving microenvironment stimulates AF cell homeostasis or remodeling.